Follow these important guidelines when transfecting DNA into CHO-K1 cells using Lipofectamine® LTX Reagent:

The addition of antibiotics to media during transfection may result in cell death. If you wish to use antibiotics during transfection, test your conditions thoroughly.

Maintain the same seeding conditions between experiments. Use low-passage cells; make sure that cells are healthy and greater than 90% viable before transfection.

Transfection can be performed both in the presence or absence of serum. Test serum-free media for compatibility with Lipofectamine® LTX Reagent.

We recommend Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute the DNA and Lipofectamine® LTX Reagent before complexing.

Visit www.lifetechnologies.com/transfection or contact Technical Service for other specialized transfection protocols (including cell-type specific advice on use of PLUS™ Reagent and antibiotics, and a protocol for vector-based RNAi).

Lipofectamine® LTX Reagent performs well with vector-based RNAi experiments. For siRNA and Stealth™ RNAi transfections, we recommend Lipofectamine® RNAiMAX (Cat. No. 13778-075). Go to www.lifetechnologies.com/RNAi or contact Technical Service for more information.

Transfection of CHO-K1 Cells

Use this procedure to transfect plasmid DNA into CHO-K1 cells in a 24-well format (for other formats, see Scaling Up or Down Transfections, below). All amounts and volumes are given on a per well basis.

The day before transfection, trypsinize and count the cells. Plate 4 x 104 cells per well in 0.5 ml of complete growth medium. Cell density should be 50~80% confluent on the day of transfection.

For each well of cells to be transfected, dilute 0.5 μg of DNA into 100 μl of Opti-MEM® I Reduced Serum Medium without serum.

For each well of cells, dilute 0.75-2.75 μl of Lipofectamine® LTX into the above diluted DNA solution, mix gently and incubate for 25 minutes at room temperature to form DNA-Lipofectamine® LTX complexes.

Remove growth medium from cells and replace with 0.5 ml of complete growth medium. Add 100 μl of the DNA-Lipofectamine® LTX complexes directly to each well containing cells and mix gently by rocking the plate back and forth.

Complexes do not have to be removed following transfection. Incubate the cells at 37°C in a CO2 incubator for 18-24 hours post-transfection before assaying for transgene expression.

Scaling Up or Down Transfections

To transfect CHO-K1 cells in different tissue culture formats, vary the amounts of Lipofectamine® LTX Reagent, DNA, cells, medium and PLUS™ Reagent used in proportion to the relative surface area, as shown in the table (amounts given on a per well basis).

Culture vessel

Surface
area per
well1

Volume plating medium

Cells per well

Volume
dilution
medium2

DNA

Lipofectamine®
LTX Reagent

96-well

0.3 cm2

100 μl

8 x 103

20 μl

100 ng

0.15 - 0.55 μl

48-well

1 cm2

200 μl

2 x 104

40 μl

200 ng

0.3 - 1.1 μl

24-well

2 cm2

500 μl

4 x 104

100 μl

500 ng

0.75 - 2.75 μl

12-well

4 cm2

1 ml

8 x 104

200 μl

1 μg

1.5 - 5.5 μl

6-well

10 cm2

2 ml

2 x 105

500 μl

2.5 μg

3.75 - 13.75 μl

1 Surface areas may vary depending on the manufacturer.
2 If the volume of Lipofectamine® LTX Reagent is too small to dispense accurately, and you cannot pool dilutions, predilute Lipofectamine® LTX Reagent 10-fold in Opti-MEM® I Reduced Serum Medium, and dispense a 10-fold higher amount (should be at least 1.0 μl per well). Discard any unused diluted Lipofectamine® LTX Reagent.